This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating G protein-coupled receptor (GPCR) signal transduction and to elucidate the mechanism of action of ethanol in these systems. GPCRs are ubiquitous components of signal transduction pathways, including taste, smell, vision, and many neurotransmitter systems. GPCRs are also targets of a great many pharmaceutical drugs. The visual transduction pathway of the retinal rod photoreceptor is the best characterized member of this receptor superfamily and is being used as a model system in these studies. Three projects were initiated this year; 1) A comparison of the functional properties of membrane proteins reconstituted with two different methods, 2) An investigation of the effect of membrane composition on GPCR stability, 3) Determination of whether or not the GPCR rhodopsin exists a s a dimer or monomer in its native membrane.[unreadable] Reconstituting membrane proteins into lipid vesicles is a useful tool for studying lipid-protein interactions as well as membrane protein function. Only a few methods, such as detergent dialysis and rapid dilution methods are widely used in studies where restored protein function is required. However, direct comparison of protein functionality between vesicles prepared by these methods is rarely available. We reconstituted in vesicles prepared by dialysis and dilution methods. The vesicles were fractionated on 0-50% sucrose density gradients. In order to assess rhodopsin function we compared the kinetics and equilibrium concentration of metarhodopsin II, the active form of light-stimulated rhodopsin that binds and activates transducin. The dialysis method yielded two pools of lipid vesicles, but only one contained rhodopsin. Varying lipid-to-rhodopsin ratio in the starting mixed micelles shifted the size of the lipid pool and only slightly changed the lipid-to-protein ratio in the rhodopsin-containing vesicles prepared by dialysis. The rapid dilution method yielded one lipid-containing band in the sucrose gradient and the lipid-to-protein ratio in the starting mixed micelles was maintained in the final vesicles. The kinetics of MII formation was greatly reduced in vesicles prepared by the dialysis method, while the activation energy for MI-MII transition remained unchanged. The yield of MII formation was also reduced in vesicles produced by the dialysis method. These observations are partially explained by the differences in lipid-to-protein ratios in these preparations, consistent with our previous finding. The sizes of the vesicles were also different based on light scattering measurement, which may also contribute to the functional differences. Overall, this study showed that while both methods produced functional rhodopsin vesicles, the composition and morphology of the vesicles were different, which lead to changes in rhodopsin activity. [unreadable] Previous studies of the effect of rhodopsin packing density in reconstituted membranes demonstrate that the thermal stability of rhodopsin is unchanged by increased rhodopsin packing density, even while membrane dynamics are dramatically altered. Given that membrane dynamics are expected to impact the unfolding of membrane proteins, we have further investigated the effects of membrane dynamics on the thermal and kinetic stability of rhodopsin. To determine the effect of membrane composition on the kinetic stability of rhodopsin, reconstituted vesicles were prepared consisting of SOPC with and without 30% cholesterol and 80:20 SOPC/SDPC with and without 30% cholesterol at L:P ratios of 100:1. Thermograms of rhodopsin thermal denaturation were obtained at four scan rates. The activation energy (Ea) of rhodopsin was calculated by four independent methods: the dependence of Tm on scan rate, the dependence of the denaturation rate constant on temperature, the dependence of heat absorption on temperature, and the transition maximum heat capacity. The effects of lipid composition on bilayer order and rhodopsin function were determined by measuring time-resolved diphenylhexatriene (DPH) fluorescence polarization and the MetaI-MetaII equilibrium (Keq). The addition of cholesterol in both SOPC and SOPC/SDPC samples resulted in a significant increase in Ea, while the addition of SDPC to SOPC caused a significant lowering of Ea. The cholesterol-containing samples exhibited increased membrane order and lower Keq, while the SDPC containing samples had decreased membrane order and higher Keq, in agreement with all previous studies.This study demonstrates that an increase in membrane order leads to an increase in the activation energy of thermal denaturation of rhodopsin. This correlation is consistent with the concept that increased protein conformational space promotes both MetaII formation and protein unfolding.[unreadable] It is well-known that the hydrophobic environment plays an important role on the stability and function of rhodopsin. We investigated the role of the hydrophobic environment on the intermolecular interactions among rhodopsin molecules, which affects the overall organization of rhodopsin molecules in the membrane. The thermal denaturation of rhodopsin in native disk membranes, reconstituted lipid membranes, and OG micelles was studied using differential scanning calorimetry. The thermal transition temperature of rhodopsin was dependent on the hydrophobic environment. The Tm of rhodopsin denaturation was 68.5C in native disk membranes, while in OG micelles it was down-shifted by 23.7C, indicating the role of the hydrophobic environment to rhodopsin. The thermogram of rhodopsin denaturation in native disk membranes was highly cooperative as evident from a narrow transition band, while the thermal transition band of rhodopsin in reconstituted lipid membranes or detergent micelles was rather broad. Detailed analysis of rhodopsin denaturation according to the following models: monomer denaturation; dimer denaturation; and dimer dissociation, indicated that rhodopsin molecules in native disk membranes denatured as dimers, while they denatured as monomers in reconstituted lipid membranes and OG micelles. Our results suggest that rhodopsin dimer formation is dependent on the orientation of rhodopsin in the membrane, since it denatures as monomer in reconstituted membranes or OG micelles where rhodopsin molecules may be arranged in a head-to-tail orientation.